Part 1 The first part of the research plan is to assess highly attenuated recombinant poxvirus vaccine candidates in the rhesus macaque model. This work is a collaborative effort with Aventis-Pasteur, which has a CRADA with the NCI, and involves intra-agency collaboration with the NIAID. Two vectors are being tested: a canarypox-based vector (ALVAC) and the genetically attenuated vaccinia strain (a derivative of the Copenhagen strain) NYVAC. Both vectors are able to complete their replication cycle and express heterologous genes under the control of early or early/late promoters. The working hypothesis is that, because both poxvirus vectors, even in the absence of a complete life cycle, still express a large number of their proteins, the response against the heterologous antigens may be limited. Thus we have investigated the hypothesis that conventional DNA plasmid (optimized for expression) may prime immune responses that may further amplify boosting with recombinant poxviruses carrying the same SIV genes. The results obtained indicate that, indeed, priming with DNA and boosting with NYVAC-SIV potentiate the virus-specific immune response at least tenfold. Accordingly, this immunization regimen afforded better containment of viremia following challenge exposure. We are also testing the importance of the addition of all the accessory/regulatory genes to the SIV/HIV structural genes. For this purpose we have constructed a novel recombinant protein encompassing the genetic information of rev, nef, and tat devoided of active site. This recombinant antigen will be used as a vaccine component. Once an optimal combination of modalities is achieved, other regimens of immunization may be further optimized by investigating the importance of the route of immunization and the help of immunomodulatory molecules. Part 2 In view of the encouraging results obtained with a preventive NYVAC-SIV-gag-pol-env vaccine candidate in rhesus macaques, we hypothesized that such a vaccine could also be effective in a regimen of immune intervention following SIV infection. Thus, we develop a model in macaques whereby to test the effect of vaccination in the presence or absence of antiretroviral therapy. A study was designed to assess whether vaccination of macaques treated with antiretroviral therapy could enhance SIV-specific CD4+ T-helper and CD8+ T-cell cytotoxic responses and whether vaccination increased the ability of the host to maintain viremia at low levels following antiretroviral therapy suspension. The highly attenuated poxvirus NYVAC-SIV-gag-pol-env live recombinant vaccine candidate was chosen as a vaccine approach to test these concepts because of its demonstrated effectiveness as a preventive vaccine candidate in macaques. The results demonstrate the feasibility to establish an animal model in which immunization and immune intervention could be assessed in a controlled fashion and that both CD4+ and CD8+ T-cell responses were elicited/boosted by a vaccine in infected animals treated with HAART. This point is very important in the context of the present concern that HAART may silence the immune response to the detriment of the host. The use of this attenuated live vector is safe in animals with active viral replication as well as in HAART-treated macaques, suggesting that the approach may be used in humans. In this study, although a possible vaccine effect could be inferred, the small number of macaques did not allow for clear assessment of vaccine relative efficacy. Therefore, a larger study on efficacy of vaccination in chronically SIV251 infected macaques was initiated, and preliminary results are very encouraging. Because of the encouraging nature of these results, a safety and immunogenicity trial in HIV-I-infected individuals will be initiated in the year 2001 using a NYVAC-based HIV-I vaccine.